EP0181491B1 - Device for simultaneously carrying out a plurality of chemical reaction steps - Google Patents

Abstract

1. Claims for the Contracting States : BE, GB, NL, AT An apparatus for perfoming chemical reaction sequences, characterized by a first number n of reaction discs (1) which are superposed in the form of a stack and are individually displaceable relative to one another, means for defining the displacement in a second number m of identical steps, m continuous bores (11, 14) disposed in the discs at the step distances from one another, one of the bores being widened out per disc to form a reaction chamber (14), means for retaining a support material in the reaction chamber on the flow of a reagent there-through. 1. Claims for the Contracting States : DE, FR, IT, CH, LI, SE An apparatus for performing chemical reaction sequences having a first number n of reaction discs (1) which are superposed in the form of a stack and are individually displaceable relative to one another, means for defining the displacement in a second number m of identical steps, m continuous bores (11, 14) disposed in the discs at the step distances from one another, one of the bores being widened out per disc to form a reaction chamber (14), means for retaining a support material in the reaction chamber on the flow of a reagent therethrough, characterized in that the undersides of the reaction discs have grooves extending concentrically to the bores and containing O-rings.

Description

The invention relates to a device for carrying out a large number of reaction sequences. Such a device is particularly suitable for the synthesis of polymer molecules such as nucleotides, proteins etc. on a carrier material such as. B. glass, silica gel or other suitable material.

As a rule, the chemical structure of such polymer molecules takes place in reaction chambers (e.g. frits, columns, etc.) in which the first building block of the molecules to be synthesized is bound to the corresponding polymer material and the reagents required for the synthesis are metered in either manually or automatically can be done.

It is in the nature of the reaction sequences that it takes a relatively long time to build up a longer-chain molecule. If you need a large number of different polymer molecules made up of the same building blocks, the time it takes to get all the desired synthesis products is often unbearably long. This results in a need for a possibility of building up a large number of different polymer molecules from the same reagents simultaneously. In this sense, the object of the invention is to provide a device with which a large number of reaction sequences can be carried out simultaneously.

According to the invention, this was achieved by the device defined in the claim.

The device is used specifically for the simultaneous synthesis of a large number of DNA and RNA segments of different chain lengths and sequences on a polymeric carrier material. In a manner known per se, the DNA segments are built up from mononucleotides, it being possible to use any suitable carrier material, preferably glass particles and various synthesis strategies, preferably the triester or the phosphite-triester method.

• Fig. 1 eine Vorrichtung mit zehn Reaktionsscheiben in perspektivischer Ansicht, teilweise aufgeschnitten,
• Fig. 2 eine Aufsicht auf eine einzelne Reaktionsscheibe.
• Fig. 1 zeigt eine Vorrichtung zur gleichzeitigen Synthese von zehn verschiedenen DNA-Segmenten, wobei als Träger ein Polymergranulat verwendet wird. Der jeweils erste Baustein der zu synthetisierenden Ketten befindet sich vor dem Synthesebeginn bereits auf dem Träger.

An exemplary embodiment of the invention is described below with reference to the accompanying drawings. Show it

• 1 shows a device with ten reaction disks in a perspective view, partially cut away,
• Fig. 2 is a plan view of a single reaction disk.
• 1 shows a device for the simultaneous synthesis of ten different DNA segments, a polymer granulate being used as the carrier. The first building block of the chains to be synthesized is already on the carrier before the synthesis begins.

The device consists of a stack of concentric round disks in which several channel systems are formed. The so-called reaction disks 1-10 are assigned to the ten oligonucleotides to be synthesized, i. H. A specific oligonucleotide is built up in each reaction disk. For this purpose, for example, the first reaction disk 1 has a reaction chamber 11. The reaction chamber 11 is a bore located approximately halfway between the axis and the edge of the disc, extending from the top surface to approximately three quarters of the disc thickness. The chamber is used to hold the carrier granulate. At the upper edge, the chamber has a circumferential widening for receiving a frit 12, which closes off the reaction chamber 11 at the top. In a concentric recess of the chamber bottom there is a further frit 13, which seals the end of the chamber 11 downwards with respect to a continuous bore with a very small diameter, eg from the chamber bottom to the lower surface of the disk. B. forms 1 mm.

The reaction disc 1 has in addition to the reaction chamber 11 four through holes 14 of the same small diameter of z. B. 1 mm. These holes are offset from the reaction chamber by 72 °, 144 °, 216 ° and 288 °.

The other reaction disks are constructed in the same way. They can be individually rotated against each other around a central connecting element.

The immediately adjoining reaction disk 2 is shown rotated by 144 ° in the drawing, so that its reaction chamber 15 lies coaxially with the bore 14 of the first disk 1.

The next reaction disk 3, which is no longer shown cut away in the drawing, is again in the position in which its reaction chamber — recognizable by the frit 16 — is aligned with the reaction chamber 11 of the disk 1. A bore 17 offset by 72 ° can be seen in the disk 3.

The stack of reaction disks 1-10 is delimited by an upper connecting disk 18 for connecting the supply hose lines for the reagents and a lower connecting disk 21 for connecting the outgoing hose lines. The upper connecting disk 18 has bores 19, which are aligned with the channels or reaction chambers in the reaction disks, with threads for screwing in the fittings for the supply hoses. The bores 19, which extend approximately up to three quarters of the disk thickness, continue, similar to the reaction chambers, in continuous narrow bores to the underside of the plate. The lower connection disk 21 can either be the same as the upper one or it has a simple collecting channel (not shown) if the used reagents no longer have to be conducted separately.

The reaction disks 1-10 and the connecting disk 18 have the narrow bores ring-shaped grooves on their lower surfaces which are used to hold O-rings with which the channels are sealed at the transitions between the washers. These sealing systems have been omitted from the drawing for the sake of clarity.

An upper pressure plate 20 is arranged above the upper connecting plate 18. Correspondingly, a lower pressure plate 22 is located under the lower connection plate 21. These pressure plates 20, 22 transmit the force which is generated by a bolt 23 which serves as a central connecting element and which is located in an axial bore through the entire stack and is provided with a screw (nut 24) on the stack. This pressing force presses the panes together in such a way that absolute tightness of the channels is ensured and any dead space that is possible within the sealing rings is eliminated.

By appropriately aligning the reaction disks 1-10, their reaction chambers and holes are aligned in such a way that there are four channels in the stack, each of which is now loaded with one of the mononucleotides A, T, C or G (indicated by arrows in the drawing), as a result of which the growing pieces of DNA are simultaneously extended by the corresponding nucleotide.

For this purpose, the reaction chamber 11 of disk 1 is placed for the appropriate time in the channel whose nucleotide is to be attached (located in the drawing in the channel in which T is used for extension, while the chamber of disk 2 is in the C channel is located: C would be deposited here in the same time as for disc 1 T). All reactions and washing processes (deprotection, capping, possibly oxidation) required for linking the mononucleotides also take place simultaneously (continuous flow process). After a connection cycle has ended, the individual disks are rotated again so that their chambers are located in the channel of the next nucleotide to be attached. To turn the discs, the nut 24 is loosened and then tightened again. When the synthesis of a DNA fragment in one of the disks has ended, its chamber is placed in the empty position, the remaining four narrow bores of the disk maintaining the four connecting channels for the extension of the DNA segments in the other reaction disks.

After the synthesis of all DNA segments in the individual reaction disks has been completed, the carrier material with the adhering synthesized nucleotide sequence is removed from them after disassembling the device and removing the frit 12. After the protective groups have been split off and detached from the carrier material, the desired unprotected DNA segments are obtained in pure form from the raw mixtures by means of a suitable cleaning method.

In order to limit the individual rotation of the individual reaction disks to the reasonable angular steps of 72 °, at least visible markings must be attached. However, it is better to use a mechanism that locks into the correct angular position.

The device can be operated both manually and mechanically with suitable drive elements. The latter mode of operation can also be combined with program control.

The device described above as an exemplary embodiment consists of reaction disks with a diameter of approximately 60 mm and a thickness of approximately 10 mm. As already mentioned, the bores 14 have a diameter of approximately 1 mm. The reaction chambers have a diameter of approx. 6 mm. However, these dimensions are only of importance in connection with a specific synthesis program. Devices with the same design and functional principle can have selectable dimensions within wide limits and in particular can be significantly larger than the exemplary embodiment described. The number of holes per disk can also be larger than in the example described above. This is important for peptide synthesis, for example.

In addition, of course, the shape of the disks and especially the reaction chambers is in no way limited to the exemplary embodiment. For example, instead of the circular arrangement of the bores on circular disks, it would be possible to provide a linear arrangement of the bores and accordingly a linear displacement of the disks instead of the rotation.

Claims (6)

1. An apparatus for performing chemical reaction sequences having

a first number n of reaction discs (1) which are superposed in the form of a stack and are individually displaceable relative to one another,

means for defining the displacement in a second number m of identical steps,

m continuous bores (11, 14) disposed in the discs at the step distances from one another, one of the bores being widened out per disc to form a reaction chamber (14),

means for retaining a support material in the reaction chamber on the flow of a reagent therethrough, characterized in that the undersides of the reaction discs have grooves extending concentrically to the bores and containing O-rings.

2. An apparatus according to claim 1, characterized in that the bores are linearly disposed and the reaction discs are linearly displaceable relative to one another.

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